Age-Dependent Susceptibility to Pulmonary Fibrosis Is Associated with NLRP3 Inflammasome Activation

Abstract

Aging has been implicated in the development of pulmonary fibrosis, which has seen a sharp increase in incidence in those older than 50 years. Recent studies demonstrate a role for the nucleotide-binding domain and leucine rich repeat containing family, pyrin domain containing 3 (NLRP3) inflammasome and its regulated cytokines in experimental lung fibrosis. In this study, we tested the hypothesis that age-related NLRP3 inflammasome activation is an important predisposing factor in the development of pulmonary fibrosis. Briefly, young and aged wild-type and NLRP3(-/-) mice were subjected to bleomycin-induced lung injury. Pulmonary fibrosis was determined by histology and hydroxyproline accumulation. Bone marrow and alveolar macrophages were isolated from these mice. NLRP3 inflammasome activation was assessed by co-immunoprecipitation experiments. IL-1β and IL-18 production was measured by ELISA. The current study demonstrated that aged wild-type mice developed more lung fibrosis and exhibited increased morbidity and mortality after bleomycin-induced lung injury, when compared with young mice. Bleomycin-exposed aged NLRP3(-/-) mice had reduced fibrosis compared with their wild-type age-matched counterparts. Bone marrow-derived and alveolar macrophages from aged mice displayed higher levels of NLRP3 inflammasome activation and caspase-1-dependent IL-1β and IL-18 production, which was associated with altered mitochondrial function and increased production of reactive oxygen species. Our study demonstrated that age-dependent increases in alveolar macrophage mitochondrial reactive oxygen species production and NLRP3 inflammasome activation contribute to the development of experimental fibrosis.

Keywords: NLRP3 inflammasome; aging; bleomycin; mitochondrial oxidative stress; pulmonary fibrosis.

Figure 1.

Increased mortality and fibrosis in aged mice after in vivo bleomycin administration. Young (2–4 mo of age) and aged (17–19 mo of age) C57BL/6 mice were instilled with bleomycin (0.1 mg/ mouse) via oral aspiration. (A) Survival of young (n = 10) and aged (n = 10) C57BL/6 mice after bleomycin instillation (Mantel Cox test, P = 0.0015). (B) Right lobe tissue from mice exposed to a range of bleomycin doses was collected on Day 28, dehydrated overnight, and digested with 6N HCl before quantification of hydroxyproline levels. Results are presented as mean ± SEM. (C) Lungs were isolated on Day 28 after instillation, fixed, and stained with H&E or trichrome before blinded analysis by a pathologist. 20× images were obtained using the Nanozoomer Digital Pathology System. Results are representative of three independent experiments. BLM, bleomycin; H&E, hematoxylin and eosin.

Figure 2.

Enhanced transforming growth factor (TGF)-β–mediated signaling and apoptosis in aged lung in response to bleomycin. Young (2–4 mo of age) (n = 10) and aged (17–19 mo of age) (n = 10) C57BL/6 mice were instilled with bleomycin (0.1 mg/mouse) via oral aspiration. (A) Phospho-SMAD3 (p-SMAD3) and SMAD3 levels present in young and aged lung isolated on Day 16 after bleomycin instillation were determined by immunoblotting. The ratio p-SMAD3/total SMAD3 expression was determined by densitometry and is expressed as fold induction in bleomycin-treated mice compared with uninjured control mice. β-actin served as the standard. P = 0.0019 (t test). (B) Relative expression of key TGF-β signaling genes in young and aged lung on Day 16 after bleomycin instillation. Student’s t test for single genes: *P < 0.05; ***P < 0.001; for all genes: two-way analysis of variance, P < 0.0001. (C) Caspase-8 and caspase-9 expression and presence of activated cleavage products in young and aged lung 28 days after bleomycin instillation were assessed by immunoblotting. β-actin served as the standard. (D) Apoptosis was assessed by TUNEL staining. Lung tissue was costained with endothelial marker CD31. Images and immunoblots are representative of at least three experiments. Data are expressed as the mean ± SD. ****P < 0.0001, **P = 0.0019. AU, arbitrary unit; CCR2, C-C motif chemokine receptor 2; COL12, collagen type-1; Csp, caspase; ENG, endoglin; PAI, plasminogen activator inhibitor; SMAD, mothers against decapentaplegic homolog.

Figure 3.

Increased nucleotide-binding domain and leucine rich repeat containing family, pyrin domain containing 3 (NLRP3) inflammasome activation in aged lung after treatment with bleomycin. Lung tissue was isolated from saline control mice and bleomycin-instilled young (2–4 mo of age) and aged (17–19 mo of age) mice at select time points after bleomycin instillation. RNA was isolated and (A) NLRP3 and (B) ASC mRNA expression was assessed by real-time polymerase chain reaction. (C) Lung homogenate samples were isolated on Day 3 after bleomycin and co-immunoprecipitation was performed against anti-ASC. Eluted proteins were separated by electrophoresis and immunoblotted with antimouse NLRP3, ASC, and pro–caspase-1. (D) Caspase-1 activity was assessed in young and aged lung homogenates isolated on Days 3 and 7 after bleomycin instillation. Similar results were obtained from two or more independent experiments with an n ≥ 5 per experiment. Data are expressed as mean ± SEM. *P < 0.05, ***P < 0.001, Student’s t test. NLRP3: Day 3, P = 0.014 (t test); Day 7, P = 0.0134 (t test); Day 10, P = 0.0463 (t test). ASC: Day 3, P = 0.0101 (t test); Day 7, P = 0.0496 (t test); Day 10, P = 0.05 (t test); Day 16, P = 0.0402 (t test). Caspase-1: Day 3, P = 0.036 (t test); Day 7, P < 0.001 (t test). ASC, apoptosis-associated speck-like protein containing a CARD domain; IB, immunoblot; IP, immunoprecipitation.

Figure 4.

Increased production of mature IL-1β and IL-18 in aged lung after treatment with bleomycin. Lung tissue was isolated from saline control mice and bleomycin-instilled young (2–4 mo of age) and aged (17–19 mo of age) mice at select time points after instillation. RNA was isolated and mRNA expression of (A) pro–IL-1β and (B) pro–IL-18 was assessed by real-time polymerase chain reaction. Homogenates were collected, and production of (C) IL-1β and (D) IL-18 in young and aged lung was assessed by ELISA. Similar results were obtained from two or more independent experiments with an n ≥ 5 per experiment. Data are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, Student’s t test. pro–IL-1β: Day 3, P = 0.0195 (t test); Day 7, P = 0.0076 (t test); Day 10, P = 0.0044 (t test); Day 16, P = 0.0154 (t test). pro–IL-18: Day 3, P = 0.0016 (t test); Day 7, P = 0.01722 (t test). IL-1β: Day 3, P < 0.0001 (t test); Day 7, P < 0.0001 (t test). IL-18: Day 3, P < 0.0001 (t test); Day 7, P < 0.0001 (t test); Day 16, P = 0.0027 (t test).

Figure 5.

NLRP3^−/− aged mice are protected from bleomycin-induced mortality and fibrosis. Wild-type and NLRP3^−/− mice (13 mo old) were instilled with bleomycin (0.1 mg/mouse) via oral aspiration. (A) Survival of wild-type (n = 8) and NLRP3^−/− (n = 10) mice after bleomycin instillation (Mantel Cox test, P = 0.0049). (B) Right lobe tissue from mice was collected on Day 21 after bleomycin instillation, dehydrated overnight, and digested with 6N HCl before quantification of hydroxyproline levels. (C) Bronchoalveolar lavage (BAL) was performed on mice at Day 21, and cell counts were performed. Data are presented as mean ± SEM. Student’s t test, **P = 0.0022 and ****P < 0.0001.

Figure 6.

NLRP3 inflammasome–mediated expression and activity in aged alveolar and bone marrow–derived macrophages is increased in response to bleomycin. Young (2–4 mo of age) and aged (17–19 mo of age) alveolar and bone marrow–derived macrophages (BMDMs) were cultured in media alone or in media containing LPS (100 ng/ml) for 4 hours before treatment with media or media containing bleomycin (0.1 U) for 24 hours. BMDMs were obtained from bone marrow cells cultured with murine M-CSF (10 ng/ml) for 7 days in 37°C, 5% CO₂. In alveolar macrophages, production of mature (A) IL-1β (P = 0.0039, t test) and (B) IL-18 (P = 0.0052, t test) was assessed by ELISA. In BMDMs treated with LPS and bleomycin, (C) expression of NLRP3 and ASC was examined by Western blotting, (D) production of IL-1β (P = 0.0068, t test) and IL-18 (P = 0.0086, t test) was measured by ELISA, and (E) caspase-1 activity was assessed by the caspase-1 assay (P = 0.0068, t test). Similar results were obtained from three or more independent experiments with an n ≥ 3 per experiment and are expressed as mean ± SEM. **P < 0.05. M-CSF, macrophage-colony stimulating factor.

Figure 7.

Production of IL-1β and IL-18 by bleomycin-treated macrophages is NLRP3 and caspase-1 dependent. Young (2–4 mo of age) and aged (17–19 mo of age) bone marrow cells were cultured with murine M-CSF (10 ng/ml) for 7 days in 37°C, 5% CO₂. Cells were treated with (A and B) missense or NLRP3-specific small interfering (siRNA) for 24 hours before LPS (100 ng/ml, 4 h) stimulation or with (C and D) glybenclamide (25 µg/ml, 24 h) at the time of LPS stimulation. Cells were subsequently cultured with media or media containing bleomycin (0.1 U) for 24 hours. IL-1β and IL-18 production was assessed by ELISA. (A) IL-1β after siRNA: LPS + BLM treatment in young, *P = 0.022 (t test); LPS + BLM treatment in aged, **P = 0.0019 (t test). (B) IL-18 after siRNA: LPS + BLM treatment in young, *P = 0.022 (t test); LPS + BLM treatment in aged, **P = 0.0106 (t test). (C) IL-1β after glybenclamide: LPS + BLM + GLY treatment in aged, **P = 0.0055 (t test). (D) IL-18 after glybenclamide: LPS + BLM + GLY treatment in aged, **P = 0.0019 (t test). In additional experiments, at the time of bleomycin stimulation, cells were treated with z-VAD (10 µM, 1 h) before IL-1β and IL-18 measurement by ELISA. (E) IL-1β: LPS + BLM + z-VAD: ***P < 0.0001 (t test)] and (F) IL-18: LPS + BLM + z-VAD, **P = 0.0055 (t test). Similar results were obtained from three or more independent experiments with an n ≥ 3 per experiment and are expressed as mean ± SEM. Ctl, control; GLY, glybenclamide; z-VAD, Z-VADFMK.

Figure 8.

Alterations in mitochondrial function in aged macrophages after bleomycin treatment potentially contributes to increased NLRP3 inflammasome function. Young (2–4 mo of age) and aged (17–19 mo of age) bone marrow cells were cultured with M-CSF (10 ng/ml) for 7 days in 37°C, 5% CO₂. On Day 7, LPS-primed cells were cultured for 24 hours with bleomycin (0.1 U). (A) Reactive oxygen species levels within mitochondria were measured by staining cells with MitoSOX at 5 µM for 40 min at 37°C. Levels of MitoSOX fluorescence were determined by flow cytometry. Cells treated with bleomycin and untreated cells are represented by solid and dashed lines, respectively. (B–C) Young and aged LPS-primed macrophages were treated with rotenone (5 µM) 1 hour before treatment with media alone or bleomycin. Cell culture supernatants were isolated, and production of IL-1β (B, LPS + BLM + ROT, aged: P = 0.0001 [t test]) and IL-18 (C, LPS + BLM + ROT, aged: P = 0.0013 [t test]) was assessed by ELISA. (D–E) Young and aged macrophages were treated with mitoTEMPO (100 µM) 1 hour before stimulation with media alone or LPS. Cells were subsequently treated with media alone or media containing bleomycin and production of IL-1β (D, LPS + BLM + MT, aged: P = 0.0001 [t test] and IL-18 (E, LPS + BLM + MT, aged: P = 0.0001 [t test]) was assessed at 24 hours by ELISA. Similar results were obtained from three or more independent experiments with an n ≥ 4 per experiment and are expressed as mean ± SEM. **P = 0.0013 and ***P < 0.05. MT, mitoTEMPO; ROT, rotenone.